Despite many recent technological advances in the field of gene transfer, a number of technical issues continue to thwart the successful implementation of gene transfer-based clinical therapies for diseases affecting the heart, blood, and lungs. Accordingly, it is becoming increasingly important to consider the development of new methods for gene delivery which overcome the limitations of existing systems. The overall goal of this research program is to develop new retroviral vectors which are able to efficiently deliver genes directly to both proliferating and quiescent cells of the cardiovascular system in vivo, and which provide for the sustained expression of the transferred genes. The major hypothesis driving the proposed studies is that a retroviral vector system which couples the capacity for integration in quiescent cells with the ability to generate extremely high titer virus stocks will have broad application in both ex vivo and in vivo gene therapy strategies aimed at the treatment of diseases of the heart, blood, and lungs. In a first series of studies, we propose to generate safe, reliable, stable HIV-derived packaging cell lines useful for the production of large quantities of high titer helper-free lentivirus- derived vectors which can be highly concentrated. To minimize the opportunity for recombinational events involving homologous packaging and vector sequences that could lead to the emergence of either replication competent virus or the transmission of specific viral gene products, we will attempt to precisely engineer the expression of HIV gag-pol coding sequences through the use of specific non-retroviral expression elements. In parallel with the generation of stable packaging cell lines, we propose to develop hybrid HlV/MLV vectors modeled after the design of the MFG vector which can be efficiently packaged by HIV- derived components and efficiently express inserted genes. In a second series of studies, we will consider several strategies for generating predominantly MLV-based packaging cell lines which will potentially support the production of murine-based vectors able to transduce proliferating and quiescent cells. The ability of the different vectors systems to efficiently transduced proliferating and quiescent cells of the cardiovascular system will be assessed both in vitro and in vivo in studies conducted by the Vector Evaluation and Model Systems Core.